Streptococcus pneumoniae is the leading cause of morbidity and mortality in developed as well as developing countries. Globally, pneumococcal infections have been estimated to cause 1.2 million deaths per year. Despite the use of appropriate antibiotics and intensive care, the case fatality rate of pneumococcal bacteremia has not changed in the past 40 years and remains at 15 to 40%. The pneumococcal polysaccharide (PPS) vaccine is fairly effective in young adults, however, a recent study showed that this vaccine reduced the risk of bacteremia by merely 44% in persons over 65 years of age. Recently, the pneumococcal conjugate vaccine was introduced and has proven highly efficacious against invasive disease in children<5 years of age. However, several studies have shown that the conjugate vaccines do not offer an advantage in immunogenicity over the conventional PPS vaccine in adults or the elderly. The reasons for these age- related differences in pneumococcal vaccine efficacy remain to be elucidated. An increased understanding of the relationship between anti-PPS antibody molecular structure and protective capacity would greatly benefit future pneumococcal vaccine and adjuvant design. We have generated an extensive library of PPS4 and PPS14 specific VH and VL chains obtained post-immunization from young and elderly adults. In addition, we have characterized the immune response of each volunteer by ELISA and opsonophagocytic activity. In this application we wish to further define and explore the potential correlation between antibody molecular structure, avidity and functional antibody activity in an effort to increase our understanding of the human immune response to PPS. 1 .We will test the hypothesis that aging affects the B cell clonotype and frequency of somatic mutation in the immune response to pneumococcal polysaccharides and that these changes result in altered antibody avidity and functional activity. This will be accomplished by: 1A. Developing human monoclonal antibodies by isolating, sorting and expanding single PPS responding B cells in vitro. The PPS-specific VH/VL will be cloned and sequenced. These studies will characterize the effect of aging on the molecular structure of PPS4, PPS14 and PPS23F specific antibodies in terms of B cell clonotype and somatic mutation. 1B. Cloning and expressing the paired variable regions obtained in Specific Aim 1A into identical Fab expression vectors. This strategy will allow us to assess influence of VH/VL molecular structure, independent of constant region, on affinity and fine specificity. 2. We will test the hypothesis that the nature of the IgG subclass (Fc) has a significant influence on avidity and function. We will construct human antibodies of the lgG1 and lgG2 subclasses containing identical VH and VL chains and define potential differences in avidity and investigate its molecular basis.